Bottom Line:
Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov.We then developed a specific diagnostic PCR for Protochlamydia spp.When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia.

Affiliation: Center for Research on Intracellular Bacteria, Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland.

ABSTRACTUsing ameba coculture, we grew a Naegleria endosymbiont. Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov. We then developed a specific diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia. Further studies are needed to assess the role of Protochlamydia spp. in pneumonia.

Figure 2: A) Intra and inter-run reproducibility of the real-time PCR assessed on duplicate of plasmidic positive controls performed at 10-fold dilutions from 105 to 101 plasmid/μL during 6 successive runs. Standard deviations show the intra-run reproducibility of the real-time PCR. B) Plots of the cycle threshold (Ct) of first and second duplicates, showing intra-run and inter-run variability of the real-time PCR between duplicates of positive control. 95% confidence interval is shown by the dashed lines. C) Bland-Altman graph showing the difference of Ct of both duplicates according to the mean of the Ct of duplicates. The dashed line shows the 95% confidence interval (i.e., limit of agreement).

Mentions:
The analytical sensitivity was 10 copy/μL (Figure 2, panel A). Intra-run variability was good (Figure 2 panel B) with a Bland-Altman bias of 0.99 and a limit of agreement of 2.87 (Figure 2, panel A). Inter-run variability was low at high concentration, 1.12, 1.71, 0.82, 1.77 cycles for 105, 104, 103, 102 copies/μL, respectively. Inter-run variability was higher at low concentration, 4.22 cycles for 101 copies/μL (Figure 2, panel A). Analytical specificity was tested with bacterial and eukaryotic DNA (Table 2). The PCR slightly amplified DNA from R. crassificans, another Chlamydia-like organism. No cross-amplification was observed with any other bacteria or with human cells. The absence of cross-amplification of P. acanthamoebae is important because this Chlamydia-related bacteria is considered an emerging agent of pneumonia (4–6).

Figure 2: A) Intra and inter-run reproducibility of the real-time PCR assessed on duplicate of plasmidic positive controls performed at 10-fold dilutions from 105 to 101 plasmid/μL during 6 successive runs. Standard deviations show the intra-run reproducibility of the real-time PCR. B) Plots of the cycle threshold (Ct) of first and second duplicates, showing intra-run and inter-run variability of the real-time PCR between duplicates of positive control. 95% confidence interval is shown by the dashed lines. C) Bland-Altman graph showing the difference of Ct of both duplicates according to the mean of the Ct of duplicates. The dashed line shows the 95% confidence interval (i.e., limit of agreement).

Mentions:
The analytical sensitivity was 10 copy/μL (Figure 2, panel A). Intra-run variability was good (Figure 2 panel B) with a Bland-Altman bias of 0.99 and a limit of agreement of 2.87 (Figure 2, panel A). Inter-run variability was low at high concentration, 1.12, 1.71, 0.82, 1.77 cycles for 105, 104, 103, 102 copies/μL, respectively. Inter-run variability was higher at low concentration, 4.22 cycles for 101 copies/μL (Figure 2, panel A). Analytical specificity was tested with bacterial and eukaryotic DNA (Table 2). The PCR slightly amplified DNA from R. crassificans, another Chlamydia-like organism. No cross-amplification was observed with any other bacteria or with human cells. The absence of cross-amplification of P. acanthamoebae is important because this Chlamydia-related bacteria is considered an emerging agent of pneumonia (4–6).

Bottom Line:
Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov.We then developed a specific diagnostic PCR for Protochlamydia spp.When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia.

Affiliation:
Center for Research on Intracellular Bacteria, Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland.

ABSTRACTUsing ameba coculture, we grew a Naegleria endosymbiont. Phenotypic, genetic, and phylogenetic analyses supported its affiliation as Protochlamydia naegleriophila sp. nov. We then developed a specific diagnostic PCR for Protochlamydia spp. When applied to bronchoalveolar lavages, results of this PCR were positive for 1 patient with pneumonia. Further studies are needed to assess the role of Protochlamydia spp. in pneumonia.